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A Network Biology Approach to Antibiotic Action and Bacterial Defense Mechanisms
Summary: James Collins combines expertise in engineering, physics, and biology to design and build synthetic gene networks for applications in biotechnology and medicine and to reverse engineer the endogenous gene networks in bacteria that regulate their responses to antibiotics.
Remarkable progress in genomic research is leading to a complete map of the building blocks of biology. Knowledge of this map is, in turn, fueling the study of gene regulation, where proteins often regulate their own production or that of other proteins in a complex web of interactions. An important challenge in postgenomic research will be the dissection and analysis of the complex dynamic interactions involved in gene regulation, and the deduction of phenotypic cellular responses from the structure and behavior of such networks. The implications of the underlying logic of genetic networks are difficult to deduce through experimental techniques alone; successful approaches will, in many cases, involve the union of new experiments and computational modeling techniques.
We have been approaching this exciting area from two complementary perspectives—systems biology and synthetic biology. In systems biology, we have been taking a top-down approach to gene regulation, and developing and applying integrated experimental-computational techniques to reverse engineer and analyze naturally occurring gene regulatory networks. In synthetic biology, we have been taking a bottom-up approach to gene regulation, and using tools from nonlinear dynamics and molecular biology to model, design, and construct synthetic gene networks. Currently we are building on this work and using innovative systems biology and synthetic biology approaches to quantitatively characterize and analyze bacterial gene regulatory networks underlying cellular responses to antibiotics, the formation of persisters, and the emergence of resistance.
With the alarming spread of antibiotic-resistant strains of bacteria, a better understanding of the specific sequences of events leading to cell death from the wide range of bactericidal antibiotics is needed for future antibacterial drug development. To further our understanding of how bacteria respond and defend themselves against antibiotics, we need systems biology and synthetic biology approaches to discern the interplay between genes, proteins, and pathways. To help address this problem, we have developed rapid and scalable methods that enable construction of quantitative models of gene, protein and metabolite regulatory networks, using steady-state or time-series expression measurements and no prior information on the network structure or function.
Recently, we employed a systems biology approach to identify novel mechanisms that contribute to bacterial cell death upon DNA gyrase inhibition by the widely used quinolone antibiotic, norfloxacin. It is well known that gyrase inhibitors induce cell death by stimulating DNA damage, impeding lesion repair, and blocking replication processes. We performed phenotypic and microarray analyses on Escherichia coli treated with norfloxacin to identify additional contributors to cell death resulting from gyrase poisoning. In the course of this work, we discovered a novel oxidative damage, cell death pathway that involves reactive oxygen species and a breakdown in iron regulatory dynamics following DNA damage induction.
Current antimicrobial therapies, which cover a wide array of targets, fall into two general categories: bactericidal drugs that kill bacteria with an efficiency of >99.9 percent and bacteriostatic drugs that merely inhibit growth. Antibiotic drug-target interactions are well studied and fall into three classes: inhibition of DNA replication and repair (e.g., quinolones), inhibition of protein synthesis, and inhibition of cell-wall turnover. The bactericidal antibiotic killing mechanisms are currently attributed to the class-specific drug-target interactions. However, our understanding of many of the bacterial responses that occur as a consequence of the primary drug-target interaction that results in cell death remains incomplete.
Building upon our work with quinolones, we chose to investigate whether hydroxyl radical formation also contributes to antibiotic-induced cell death in bacteria among the other classes of antibiotics. We found in both Gram-negative and Gram-positive bacteria that the three major classes of bactericidal antibiotics (aminogylcosides, β-lactams, and quinolones), regardless of drug-target interaction, stimulate hydroxyl radical formation in bacteria. Furthermore, we demonstrated that hydroxyl radical generation contributes to the killing efficiency of these lethal drugs. We also showed, in contrast, that bacteriostatic drugs do not produce hydroxyl radicals. We demonstrated that all bactericidal drug classes utilize internal iron from iron-sulfur clusters to promote hydroxyl radical formation (via the Fenton reaction), and we showed that these events appear to be mediated by the tricarboxylic acid (TCA) cycle and a transient depletion of NADH. We propose that there is a common mechanism of cellular death underlying all classes of bactericidal antibiotics whereby harmful hydroxyl radicals are formed as a function of metabolic-related NADH depletion, leaching of iron from iron-sulfur clusters, and stimulation of the Fenton reaction.
Antibacterial drug design has focused on blocking essential cellular functions. This has yielded significant advances in antibacterial therapy; however, the ever-increasing prevalence of antibiotic-resistant strains has made it critical that we develop novel, more effective means of killing bacteria. Our work indicates that all three major classes of bactericidal drugs can be potentiated by targeting bacterial systems that remediate hydroxyl radical damage, including proteins involved in triggering the DNA damage response (i.e., the SOS response). As noted above, we are building on these efforts and using our systems biology approaches to map out the regulatory networks and pathways underlying bacterial responses to bactericidal antibiotics. These efforts could provide insights into common cellular death mechanisms and triggers for the different classes of bactericidal antibiotics.
Last updated January 16, 2009
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